Visualize the three-dimensional arrangement of vascular bundles using cone-beam optical tomography
--Pan, SJ; Shih, A; Liou, WS; Park, MS; Wang, G; Cheng, PC

Adopting from the methodology and algorithm used in X-ray cone-beam tomography (Wang and Cheng, Zool. Studies, 34: Sup. 1. 159-161, 1995), a cone-beam optical tomographic imaging system (Figure 1) has been developed at our laboratory to visualize the three-dimensional arrangement of vascular bundles. Leaf sheaths (Oh43) were used to test the imaging system. Tissues were fixed in 3:1 ethanol/acetic acid mixture, washed in running water, stained in Schiff reagent, dehydrated in acetone, and cleared in methyl salicylate (MS)(Cheng, MNL 69:28-29, 1995). The procedure produced a highly cleared tissue with a relatively homogeneous refractive index and minimum scattering. The vascular bundle and cell wall stained in magenta. A Kodak Wratten #25 red filter was placed in front of the light source to reduce scattering and contrast. The specimen was rotated by a stepper motor in a rectangular glass container filled with MS. Projection images were captured with a cooled CCD camera (equipped with a Kodak 1400 CCD chip) at 12-bit dynamic range at 1277x1004 pixels. In order to maximize the depth-of-focus of the close-up lens (Olympus OM 50mm macro lens), aperture setting of f/22 was used. One hundred equiangular projections were obtained. Figure 2 shows two views of a rolled-up leaf sheath (half of a leaf sheath) obtained by the CCD camera. The generalized Feldkamp cone-beam algorithm originally developed for X-ray microtomography (Wang et al., IEEE Trans. Med. Imag. 12:486-496, 1993) was used for the three-dimensional reconstruction. Figure 3 shows a three-dimensional isosurface rendering of the leaf sheath and its vascular bundles.

The cone-beam optical tomographic technique demonstrated in this article can be used for visualizing plant structures in 3D. Since a volumetric data set is obtained from the tomographic reconstruction, viewing the specimen at any arbitrary angle can be easily accomplished by using a computer with appropriate software. It is important to note that the derivation of the original X-ray tomographic reconstruction algorithm was based on the X-ray absorption properties of the specimens. No consideration regarding scattering and refraction was given. Therefore, the reconstruction assumption in our optical tomography was also based on the condition that both scattering and refraction resulting from the MS-cleared tissue are negligible. If the specimen is not cleared entirely to a glass-like appearance, the optical tomography can not be performed reliably.

This article is dedicated to Professor Dr. D. B. Walden on the occasion of his retirement after many years of continued inspiration through discussions with his students and colleagues in the fields of genetics. This project was supported in parts by the Academic Development Funds of SUNY to PCC.

Figure 1. Schematic representation of a cone-beam optical tomographic imaging system.
Figure 2 (a and b). Optical projection images of half a leaf sheath viewed at two different angles.
Figure 3. Isosurface view of the three dimensional data obtained from 100 optical projections. Note the vascular bundles within the leaf sheath.

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